CN101787376B - Paecilomyces varioti metabolite and application thereof - Google Patents
Paecilomyces varioti metabolite and application thereof Download PDFInfo
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- CN101787376B CN101787376B CN2010100391098A CN201010039109A CN101787376B CN 101787376 B CN101787376 B CN 101787376B CN 2010100391098 A CN2010100391098 A CN 2010100391098A CN 201010039109 A CN201010039109 A CN 201010039109A CN 101787376 B CN101787376 B CN 101787376B
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- 241001236817 Paecilomyces <Clavicipitaceae> Species 0.000 title claims abstract description 22
- 239000002207 metabolite Substances 0.000 title claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 241000684698 Paecilomyces sp. (in: Hypocreales) Species 0.000 claims abstract description 7
- 239000000575 pesticide Substances 0.000 claims abstract description 6
- 238000004519 manufacturing process Methods 0.000 claims abstract description 5
- 231100000572 poisoning Toxicity 0.000 claims abstract description 4
- 230000000607 poisoning effect Effects 0.000 claims abstract description 4
- 241000529915 Xylophilus Species 0.000 claims abstract description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 24
- 241000243771 Bursaphelenchus xylophilus Species 0.000 claims description 22
- 239000004480 active ingredient Substances 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000000499 gel Substances 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 229960001866 silicon dioxide Drugs 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 235000008331 Pinus X rigitaeda Nutrition 0.000 abstract description 6
- 235000011613 Pinus brutia Nutrition 0.000 abstract description 6
- 241000018646 Pinus brutia Species 0.000 abstract description 6
- 238000000855 fermentation Methods 0.000 abstract description 4
- 230000004151 fermentation Effects 0.000 abstract description 4
- 241000238631 Hexapoda Species 0.000 abstract description 3
- 231100000053 low toxicity Toxicity 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 241000243770 Bursaphelenchus Species 0.000 abstract 2
- 238000004090 dissolution Methods 0.000 abstract 1
- 238000007877 drug screening Methods 0.000 abstract 1
- 231100000225 lethality Toxicity 0.000 description 11
- 241000244206 Nematoda Species 0.000 description 8
- 239000000523 sample Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 231100000331 toxic Toxicity 0.000 description 5
- 230000002588 toxic effect Effects 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 4
- 241001481710 Cerambycidae Species 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 241000123650 Botrytis cinerea Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241001442207 Monochamus alternatus Species 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000000443 biocontrol Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000000855 fungicidal effect Effects 0.000 description 2
- 239000000417 fungicide Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000254173 Coleoptera Species 0.000 description 1
- 235000007466 Corylus avellana Nutrition 0.000 description 1
- 240000003211 Corylus maxima Species 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000258937 Hemiptera Species 0.000 description 1
- 235000001018 Hibiscus sabdariffa Nutrition 0.000 description 1
- 241000257303 Hymenoptera Species 0.000 description 1
- 241000188153 Isaria fumosorosea Species 0.000 description 1
- 239000005908 Isaria fumosorosea Apopka strain 97 (formely Paecilomyces fumosoroseus) Substances 0.000 description 1
- 241000243785 Meloidogyne javanica Species 0.000 description 1
- 235000011609 Pinus massoniana Nutrition 0.000 description 1
- 241000018650 Pinus massoniana Species 0.000 description 1
- 241001465752 Purpureocillium lilacinum Species 0.000 description 1
- 235000005291 Rumex acetosa Nutrition 0.000 description 1
- 240000007001 Rumex acetosella Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 238000009629 microbiological culture Methods 0.000 description 1
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- 230000003071 parasitic effect Effects 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
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- 238000007665 sagging Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 235000003513 sheep sorrel Nutrition 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 239000011122 softwood Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
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Abstract
The invention relates to a paecilomyces metabolite and application thereof, belonging to the technical field of microbial drug screening. The invention is realized by the following steps: the production strain is Paecilomyces sp.1788 with the deposit number: CGMCC No. 3422; paecilomyces sp.1788 is cultured in Chachi culture medium through conventional liquid fermentation, the fermented liquid is concentrated at low pressure, and the water phase is eluted to obtain the metabolite of Paecilomyces. The paecilomyces metabolite has the function of poisoning pine wood nematodes (Bursaphelenchuchuh xylophilus)Semi-lethality (ED) at 24 hours50) 299.5ppm, ED95Was 796.2 ppm. Can be used for preparing a preparation for killing pine wood nematodes. Compared with the existing pesticide for killing pine thread insects, the pesticide has the characteristics of high efficiency, low toxicity, easy water dissolution, easy preparation and the like.
Description
Technical field:
The present invention relates to a kind of Paecilomyces varioti meta-bolites and application thereof, belong to microbial medicine triage techniques field.
Background technology:
Some kinds in the paecilomyces play an important role in the controlling plant insect pest; Like the control of Paecilomyces lilacinus to plant nematodes such as SCN, root knot nematodes; Parasitic and the various insects that causes death of paecilomyces fumosoroseus and powder Paecilomyces varioti ability comprises Homoptera, lepidopteran, Diptera, Coleoptera, Hymenoptera etc.In many countries, these Paecilomyces variotis successfully are developed to biocontrol fungicide, in making up the ecological agriculture, play an important role.
Paecilomyces (Paecilomyces Bainier) belongs to Deuteromycotina (Deuteromycotina), hyphomycetes (Hyphomycetes), hyphomycetales (Hyphomycetales), Moniliaceae (Moniliaceae).Main morphological specificity is the mycelia multinuclear, and white, pearl, spread from the hair shape to dark white or brown.Conidiophore produces from aerial hyphae or the funiculus that spreads, but many kind lacks clear and definite sporophore.Have or do not have coremium to form, length variations is very big.Bottle metulae portion's column or sphere are expanded, and upwards spindle often surpasses the half the of length overall, many crooked main shafts that also depart from; It is complicated that bottle is obstructing the situation of giving birth to, or be the branch of colyliform as mould, or grow thickly brokenly on short and small branch, or singly be born on the aerial hyphae that spreads.The conidium unit cell, the column of pseudovum circle, ellipse, kidney shape or spindle, the minority kind is subsphaeroidal.Conidium is gathered into dry discrete chain usually, sometimes oblique arrangement or form irregular spore agglomerate.On the Cha Shi substratum, bacterium colony is white, pearl, greenish orange yellow, yellowish pink, fuzzy filbert, tawny or real green usually.
Pine wood nematode nineteen eighty-two finds in the Zhongshan Tomb, Nanjing first, later in succession in Jiangsu, Anhui, Guangdong and zhejiang and other places cause disaster, and almost destroyed the masson pine forest that extensively distributes in Hong Kong.Closely propagate the main media longicorn that leans on,, carry propagation like Monochamus alternatus (Monochamus alternatus); Mainly nursery stock, pine, pine tr and the pine goods etc. of allocation and transportation band epidemic disease (longicorn of band pine wood nematode) are propagated artificially at a distance.By the metainfective pine tree of pine wood nematode, needle tawny or sorrel, wilting are sagging, and the resin secretion stops, and on trunk, can be observed longicorn and invade the hole or the vestige of laying eggs, and the whole strain of sick tree is withered dead, the timber blue stain.The serious threat Timber stands.Because expansion rapidly, the natural softwood forest of scenic spot such as Mount Huang, Zhangjiajie has been constituted grave danger at present.Pine nematode also all has generation in states such as the U.S., Canada, Mexico, Japan, Korea S, Korea.Therefore, the biological prevention and control agent of developing to pine wood nematode seems particularly important.
Summary of the invention:
The object of the present invention is to provide a kind of Paecilomyces varioti meta-bolites, for exploitation pine wood nematode biocontrol fungicide lays the foundation.
The fungi that the present invention uses was Paecilomyces varioti 1788 (Paecilomyces sp.1788), and 1788 bacterial strains have been stored in China Microbial Culture Preservation Commission common micro-organisms center, address: China on November 5th, 2009. Beijing. and the Zhong Guan-cun; Preserving number: CGMCC No.3422.
The present invention is achieved in that Paecilomyces varioti (Paecilomyces sp.1788) through Cha Shi substratum conventional liq fermentation culture; After fermented liquid low pressure concentrated; The water warp is column chromatography repeatedly; Wash-out obtains Paecilomyces varioti meta-bolites of the present invention, and this Paecilomyces varioti meta-bolites is obtained by following steps:
A. producing bacterial strain is Paecilomyces sp.1788, preserving number: CGMCC No.3422;
B. the mycelium with 1788 bacterial strains is inoculated on the test tube PDA culture medium slant, cultivates 4-8 days down, obtains the test tube kind for 20-30 ℃;
C. the test tube kind is inoculated in the 250ml triangular flask of every bottled 50ml liquid nutrient medium, culture medium prescription is: peptone 10g, yeast extract paste 10g, glucose 20g, tap water 1000ml, pH nature; Culture temperature 20-30 ℃; Shaking table is cultivated, and rotating speed is 100-250rpm; Incubation time 5-15 days;
After the fermented liquid low pressure that d. will contain mycelia concentrates, extract the concentrated formation of the water position low pressure after extraction liquid concentrator successively with ETHYLE ACETATE and propyl carbinol; Also constantly stir in the acetone with 2 times of volumes of its slow adding; Leave standstill then, form water-soluble phase of acetone and precipitated phase, separate two phases again;
E. it is water-soluble colourless basically mutually until acetone to repeat several, the collecting precipitation phase; Precipitated phase repeated the polycaproamide post; With acetone and water gradient elution, pass through C-18 silicagel column and SephadexLH-20 gel column methanol-eluted fractions again, serve as with reference to merging component with the TLC plate; The active tracking detected institute and separated and obtain active ingredient, develops the color redness with 0.5% ethanol solution of ninhydrin.
Experiment shows: the tunning pine wood nematode poisoning of Paecilomyces varioti 1788 (Bursaphelenchuh xylophilus), 24 hours semilethal rate (ED
50) be 299.5 ppm, ED
95Be 796.2ppm.
Paecilomyces varioti meta-bolites of the present invention has the function of pine wood nematode poisoning (Bursaphelenchuhxylophilus), can kill the application of pine wood nematode preparation as preparation.
The present invention compares with the existing pine wood nematode medicine that kills, have efficient, low toxicity, soluble in water, be prone to characteristics such as preparation.
Embodiment:
The fermentation method for producing of active substance of the present invention:
1, the mycelium with 1788 bacterial strains is inoculated on the test tube culture medium slant, and culture medium prescription is the PDA substratum, cultivates 4 or 6 or 8 days down, obtains the test tube kind for 20 or 25 or 30 ℃;
2, the test tube kind is inoculated in the 250ml triangular flask in (every bottled 50ml) liquid nutrient medium, culture medium prescription is: peptone 10g, yeast extract paste 10g, glucose 20g, tap water 1000ml, pH nature.Culture temperature 20 or 25 or 30 ℃; Shaking table is cultivated, and rotating speed is 100-250 rpm; Incubation time 5 or 10 or 15 days.
After the fermented liquid low pressure that 3, will contain mycelia concentrates, extract the concentrated formation of the water position low pressure after extraction liquid concentrator successively with ETHYLE ACETATE and propyl carbinol; Also constantly stir in the acetone with 2 times of volumes of its slow adding; Leave standstill then, form water-soluble phase of acetone and precipitated phase, separate two phases again.It is water-soluble colourless basically mutually until acetone to repeat several, the collecting precipitation phase.Precipitated phase repeated the polycaproamide post; With acetone and water gradient elution, pass through C-18 silicagel column and SephadexLH-20 gel column methanol-eluted fractions again, serve as with reference to merging component with the TLC plate; The active tracking detected the active ingredient that is separated to, and develops the color red with 0.5% ethanol solution of ninhydrin.
Active ingredient of the present invention is to the test of pesticide effectiveness of pine wood nematode
1, test with medicament:
Use the sterile distilled water dilution for being suitable for concentration active ingredient.
Use the sterile distilled water dilution for being suitable for concentration nonvaccinated fermention medium as contrast.
2, nematode is used in the preparation test
(Botrytis cinerea) is connected on the PDA flat board with Botrytis cinerea; 25 ℃ are cultured to and cover with flat board, insert a substratum that has pine wood nematode (B.xylophilus), cultivate mycelia for 25 ℃ and disappear; Nematode is covered with flat board, and it is subsequent use to place 4 ℃ of refrigerators to preserve then.Wash out by the graceful funnel method of shellfish during use, regulate the nematode suspension to about 300/ml with sterilized water.
3, TP
Get 2ml specimen or control sample in the petridish of 6cm diameter, add the nematode suspension (about 100) of 0.3ml with liquid-transfering gun.When measuring activity, under anatomical lens, observe in different time (12h and 24h), record dead wire borer population (motionless with physical stimulation is standard) and total nematode number, calculation sample is to nematode " sample lethality rate " and " contrast mortality ratio ".A sample is established two repetitions, obtains " the correction lethality rate " of this bacterial strain at last.The correction lethality rate of strains tested calculates by following formula:
Sample average lethality rate (%)=∑ (the bus borer population that dead wire borer population/observation that observation count down to count down to) * 100%/n;
Proofread and correct lethality rate (%)=sample average lethality rate (%)-contrast average lethality rate (%)
Embodiment 1: Paecilomyces varioti 1788 fermented liquids are to the toxic action of pine wood nematode
Cultured liquid fermentate after with vacuum filtration clear solution, this solution is evaporated to dried active substance at 60-70 ℃, is dissolved in the sample that sterile distilled water is mixed with 5mg/ml, carries out the test of pesticide effectiveness by above-mentioned TP.Tabulation is depicted as corrected mortality.
Table 1 fermented liquid is to the toxic action of B.xylophilus nematode
The result shows that the liquid fermentation production of Paecilomyces varioti 1788 has significant toxic action to pine wood nematode, and lethality rate reaches more than 92%.
Embodiment 2: active ingredient is to the toxic action of pine wood nematode
The active ingredient that separation is obtained is dissolved in sterile distilled water, dilutes the solution for 500ppm.
Carry out the test of pesticide effectiveness by above-mentioned TP.Tabulation is depicted as corrected mortality.
Table 2 active ingredient is to the toxic action of B.xylophilus nematode
The result shows that the active ingredient that separation obtains reaches more than 80% at 24 hours the lethality rate of B.xylophilus nematode.Has tangible eelworm-killing activity.
Embodiment 3: active ingredient is to the semilethal rate ED of pine wood nematode
50
Embodiment 3 methods are with embodiment 2, and difference is: the 2.5mg active ingredient is dissolved in small amount of aseptic zero(ppm) water, is formulated as the aqueous solution of 1000ppm, semilethal rate (ED is measured in sesquialter dilution subsequently
50) and 95% lethality rate (ED
95).
The ED of table 3 pair B.xylophilus nematode
50And ED
95
The result shows that active ingredient was at 24 hours semilethal rate (ED to the B.xylophilus nematode
50) for being 299.5 ppm, 95% lethality rate (ED
95) be 796.2 ppm.
Claims (3)
1. the working method of a Paecilomyces varioti meta-bolites is characterized in that the step of this working method is following:
A. producing bacterial strain is Paecilomyces sp.1788, preserving number: CGMCC No.3422;
B. the mycelium with 1788 bacterial strains is inoculated on the test tube PDA culture medium slant, cultivates 4-8 days down, obtains the test tube kind for 20-30 ℃;
C. the test tube kind is inoculated in the 250ml triangular flask of every bottled 50ml liquid nutrient medium, culture medium prescription is: peptone 10g, yeast extract paste 10g, glucose 20g, tap water 1000ml, pH nature; Culture temperature 20-30 ℃; Shaking table is cultivated, and rotating speed is 100-250rpm; Incubation time 5-15 days;
After the fermented liquid low pressure that d. will contain mycelia concentrates, extract the concentrated formation of the water position low pressure after extraction liquid concentrator successively with ETHYLE ACETATE and propyl carbinol; Also constantly stir in the acetone with 2 times of volumes of its slow adding; Leave standstill then, form water-soluble phase of acetone and precipitated phase, separate two phases again;
E. it is water-soluble colourless basically mutually until acetone to repeat several, the collecting precipitation phase; Precipitated phase repeated the polycaproamide post; With acetone and water gradient elution, pass through C-18 silicagel column and SephadexLH-20 gel column methanol-eluted fractions again, serve as with reference to merging component with the TLC plate; The active tracking detected institute and separated and obtain active ingredient, develops the color redness with 0.5% ethanol solution of ninhydrin.
2. production bacterial strain Paecilomyces sp.1788 who produces the Paecilomyces varioti meta-bolites, preserving number: CGMCC No.3422.
3. the production bacterial strain of claim 2 has the application in the pesticide preparation of function of pine wood nematode poisoning (Bursaphelenchuh xylophilus) in preparation.
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| CN2010100391098A CN101787376B (en) | 2010-01-05 | 2010-01-05 | Paecilomyces varioti metabolite and application thereof |
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|---|---|---|---|
| CN2010100391098A CN101787376B (en) | 2010-01-05 | 2010-01-05 | Paecilomyces varioti metabolite and application thereof |
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| CN101787376B true CN101787376B (en) | 2012-05-23 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1422292A1 (en) * | 2001-08-17 | 2004-05-26 | Hangzhou Tsbio Science & Technology Co., Ltd. | Fermentation product of cyptoporus volvatus and its preparation method and use |
| CN1827633A (en) * | 2004-10-30 | 2006-09-06 | 浙江省农业科学院 | Method for extracting N6-(2-hydroxyethyl)adenosine from Paecilomyces cicadae culture |
| CN101041809A (en) * | 2007-02-14 | 2007-09-26 | 贵州大学 | Anti-drought ultraviolet resistant long-storage cicadae penicillium notatum strain and application thereof |
-
2010
- 2010-01-05 CN CN2010100391098A patent/CN101787376B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1422292A1 (en) * | 2001-08-17 | 2004-05-26 | Hangzhou Tsbio Science & Technology Co., Ltd. | Fermentation product of cyptoporus volvatus and its preparation method and use |
| CN1827633A (en) * | 2004-10-30 | 2006-09-06 | 浙江省农业科学院 | Method for extracting N6-(2-hydroxyethyl)adenosine from Paecilomyces cicadae culture |
| CN101041809A (en) * | 2007-02-14 | 2007-09-26 | 贵州大学 | Anti-drought ultraviolet resistant long-storage cicadae penicillium notatum strain and application thereof |
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